Project summary/abstract Mosquito control remains the primary tool for combating many mosquito-vectored diseases, such as Dengue, Chikungunya, Zika, and West Nile, because no approved vaccine, therapeutant or prophylaxis is currently available for use. Key deficiencies of existing vector control methods include resistance to existing insecticides and the inability to find and treat cryptic breeding sites, which provide refuge to immature mosquitoes and allow for the rapid recovery of mosquito populations following conventional control efforts. The self-delivering method known as `auto-dissemination' addresses this deficiency by using mosquitoes as the vehicle to deliver a potent mosquito growth regulator to cryptic breeding sites. The Phase 1 results have included pilot laboratory and field work, as a proof-of-principle against both Aedes aegypti and Aedes albopictus. The epidemiology of Zika has led to an urgent need for preemptive vector control approaches. Specifically, a majority of Zika virus infections in humans are not detected quickly. Delayed detection can undermine a public health protection plan that is based on reactive approaches, i.e., a strategy of initiating intensive vector control only after the detection of an epidemic, because by the time of detection, a large proportion of the human population can be infected already. The approach being developed in the proposed work is ideal as a preemptive approach, because (1) it requires small amounts of chemical, i.e., less environmental impact and (2) the artificially-reared mosquito carriers can be delivered at any time, e.g., before an indigenous vector population reaches dangerous levels. The ?Auto-Dissemination Augmented by Males? (ADAM) is based on mass-producing male mosquitoes, which do not bite or transmit pathogens. The ADAM method can utilize wild type mosquitoes, Wolbachia- infected mosquitoes or Genetically Modified mosquitoes. Integration with existing mosquito mass rearing programs can speed the uptake of the ADAM technology. Adult ADAM males are treated with an Insect Growth Regulator (IGR) and then released to deliver lethal doses of the IGR to oviposition sites. Phase I work included: 1) laboratory development, 2) regulatory work, e.g., approval of a Research Authorization from the California Department of Pesticide Regulation, 3) field trials in KY against Ae. albopictus, and 4) field trials in collaboration with the Consolidated Mosquito Abatement District (CMAD) to test the ADAM approach against Ae. aegypti. The laboratory results show that Ae. albopictus, Ae. aegypti, and Culex pipiens males can: 1) tolerate the IGR with negligible cost to the male carriers, 2) directly deliver larvicide to breeding sites, and 3) cross-contaminate females with IGR doses that are subsequently lethal to larvae. Field trials with both Ae. aegypti and Ae. albopictus show the ADAM method to cause significant larval mortality and significant reductions in the adult population, when compared to the non-treated sites. Phase 1 work also included an improvement of methods for the mass manufacturing of adult male mosquitoes. In the proposed Phase II work, we will conduct work to replicate and expand field trials against Ae. aegypti and Ae. albopictus, from proof-of-principle trials to a scale that is adequate for operational use by mosquito abatement districts. By performing field trials with multiple abatement districts from different states, our proposed Phase II work will satisfy an EPA requirement for commercial registration, i.e., that efficacy and safety be demonstrated in multiple ecological contexts. In addition to the EPA, the data will be submitted to the World Health Organization (WHO) Vector Control Advisory Group (VCAG), which serves as an advisory body on new forms of vector control. WHO approval is anticipated to increase international uptake of the ADAM technology. Additional Phase II work will extend Phase I work with Cx. pipiens, an important vector of West Nile Virus. Specifically, one or more field trials will provide efficacy data for use against this additional, important mosquito species, which is another focus of abatement districts' vector control efforts.